FIELD

[001] Biologic tissues and cells are affected by electrical stimulus. Accordingly, devices and techniques for applying electric stimulus to tissue have been developed to address a number of medical issues. The present Specification relates to methods and devices useful applying electric fields to the body, or a portion thereof such as a bodily fluid.

BACKGROUND

[002] Antibiotics are medicines used to prevent and treat bacterial infections. Antibiotic resistance occurs when bacteria change in response to the use of these medicines. These bacteria may infect humans and animals, and the infections they cause are harder to treat than those caused by non-resistant bacteria. Antibiotic resistance leads to higher medical costs, prolonged hospital stays, and increased mortality.

[003] Antibiotic resistance is rising to dangerously high levels in all parts of the world. New resistance mechanisms are emerging and spreading globally, threatening our ability to treat common infectious diseases. A growing list of infections- such as pneumonia, tuberculosis, blood poisoning, gonorrhea, and foodbome diseases- are becoming harder, and sometimes impossible, to treat as antibiotics become less effective.

[004] Where antibiotics can be bought for human or animal use without a prescription, the emergence and spread of resistance is made worse. Similarly, in countries without standard treatment guidelines, antibiotics are often over-prescribed by health workers and veterinarians and over-used by the public.

SUMMARY

[005] Aspects disclosed herein include systems, devices, and methods for preventing, limiting, reducing, or eliminating antibiotic resistance, for example using bioelectric devices that comprise a multi-array matrix of biocompatible microcells to apply an electric field, an electric current, or both to the body of, or a tissue or bodily fluid of, for example, a mammal. Alternatively, an electric field can be applied systemically to the body or a portion thereof to prevent, limit, reduce, or eliminate antibiotic resistance. For example, an electric field can be applied to the body in its entirety, or a portion thereof, or to a body fluid such as blood, which can be removed from a patient, passed through an electric field and/or current, and returned to a patient.

[006] Disclosed embodiments can be used to treat bacterial infections by preventing, limiting, reducing, or eliminating antibiotic resistance. For example, disclosed methods and systems can be used to treat sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[007] FIG. 1 is a detailed plan view of an embodiment disclosed herein.

[008] FIG. 2 is a detailed plan view of a pattern of applied electrical conductors in accordance with an embodiment disclosed herein.

[009] FIG. 3 is an adhesive bandage using the applied pattern of FIG. 2.

[010] FIG. 4 is a cross-section of FIG. 3 through line 3-3.

[011] FIG. 5 is a detailed plan view of an alternate embodiment disclosed herein which includes fine lines of conductive metal solution connecting electrodes.

[012] FIG. 6 is a detailed plan view of another alternate embodiment having a line pattern and dot pattern.

[013] FIG. 7 is a detailed plan view of yet another alternate embodiment having two line patterns.

[014] FIG. 8 depicts alternate embodiments showing the location of discontinuous regions as well as anchor regions of the wound management system.

[015] FIG. 9 (A) is an Energy Dispersive X-ray Spectroscopy (EDS) analysis of Ag/Zn BED (bioelectric device; refers to an embodiment as disclosed herein). A. Scanning Electron Microscope (SEM) image; B. Light Microscope Image; C and D. Closer view of a golden dot and a grey dot in B respectively. E. EDS element map of Zinc; F. EDS element map of Silver; G. EDS element map of Oxygen; H. EDS element map of Carbon. Scale bar A-B, E-H: 1 mm; C-D: 250mm (B.C) Absorbance measurement on treating planktonic PAO1 culture with placebo, Ag/Zn BED and placebo + Ag dressing and CFU measurement. (D) Zone of inhibition with placebo, Ag/Zn BED and placebo + Ag dressing.

[016] FIG. 10 depicts Scanning Electron Microscope images of in-vitro PAO1 biofilm treated with placebo, an embodiment disclosed herein ("BED"), and placebo + Ag dressing.

[017] FIG. 11 shows EPS staining.

[018] FIG. 12 shows Live/Dead staining. The green fluorescence indicates live PAO1 bacteria while the red fluorescence indicates dead bacteria.

[019] FIG. 13 shows PAO1 staining.

[020] FIG. 14 depicts Real-time PCR to assess quorum sensing gene expression.

[021] FIG. 15 shows EPR spectra using DEPMPO. Spin adduct generation upon exposure to disclosed embodiments for 40 minutes in PBS.

[022] FIG. 16 depicts Real-time PCR performed to assess mex gene expression on treatment with Ag/Zn BED and 10mM DTT.

[023] FIG. 17 shows Glycerol-3-Phosphate Dehydrogenase enzyme activity. A. OD was measured in the kinetic mode. B. GPDH activity was calculated using the formula, Glycerol- 3-Phosphate dehydrogenase activity = B/(DT X V) x Dilution Factor = nmol/ min/ml, where: B = NADH amount from Standard Curve (nmol). DT= reaction time (min). V= sample volume added into the reaction well (ml).

[024] FIG. 18 shows the effect of a disclosed embodiment on Acinetobacter baumannii.

[025] FIG. 19 shows the effect of a disclosed embodiment on Pseudomonas aeruginosa.

[026] FIG. 20 shows the effect of a disclosed embodiment on Staphylococcus aureus.

[027] FIG. 21 shows the effect of a disclosed embodiment on Klebsiella pneumoniae.

[028] FIG. 22 shows the effect of a disclosed embodiment on Klebsiella pneumoniae.

[029] FIG. 23 shows the effect of a disclosed embodiment on Acinetobacter Baumannii.

DETAILED DESCRIPTION

[030] Embodiments disclosed herein include systems that can provide a low level electric field (LLEF) to a tissue or organism (thus a "LLEF system") or, when brought into contact with an electrically conducting material, can provide a low level micro-current (LLMC) to a tissue or organism (thus a "LLMC system”). Thus, in embodiments a LLMC system is a LLEF system that is in contact with an electrically conducting material. In certain embodiments, the micro- current or electric field can be modulated, for example, to alter the duration, size, shape, field depth, current, polarity, or voltage of the system. In embodiments the watt-density of the system can be modulated. In embodiments the frequency, phase, amplitude, and wave form can be modulated. Embodiments disclosed herein comprise patterns of microcells or reservoirs or dots. The patterns can be designed to produce an electric field, an electric current, or both. In embodiments the pattern can be designed to produce a specific size, strength, density, shape, or duration of electric field or electric current. In embodiments reservoir or dot size and separation can be altered.

[031] Embodiments disclosed herein comprise biocompatible electrodes or reservoirs or dots on a surface, for example a fabric or the like. In embodiments the surface can be pliable. In embodiments the surface can comprise a gauze or mesh. Suitable types of pliable surfaces for use in embodiments disclosed herein can be cloth, absorbent textiles, low-adhesives, vapor permeable films, hydrocolloids, hydrogels, alginates, foams, foam-based materials, cellulose-based materials including Kettenbach fibers, hollow tubes, fibrous materials, such as those impregnated with anhydrous / hygroscopic materials, beads and the like, or any suitable material as known in the art. Embodiments can include coatings on the surface, such as, for example, over or between the electrodes. Such coatings can include, for example, silicone, and electrolytic mixture, hypoallergenic agents, drugs, biologies, stem cells, skin substitutes, blood coagulants or anti-coagulants, or the like. [032] In embodiments the system comprises a component such as elastic to maintain or help maintain its position. In embodiments the system comprises a component such as an adhesive to maintain or help maintain its position.

[033] Further embodiments comprise systems and devices suitable for applying an electric field to a body, for example a mammalian body. Further embodiments comprise systems and devices suitable for applying an electric field to a portion of a body, for example a mammalian body.

[034] Embodiments comprise devices for applying an electric field or current or both to a bodily fluid, for example by removing the fluid from the body, applying the field or current, for example by passing the fluid between or over/under electrodes that can establish a field or current, then returning the fluid to the body.

[036] "Activation gel” as used herein means a composition useful for maintaining a moist environment about the area to be treated or improving conductance in the area to be treated.

[036] "Affixing" as used herein can mean contacting a patient or tissue with a device or system disclosed herein.

[037] "Applied" or "apply" as used herein refers to contacting a surface with a conductive material, for example printing, painting, or spraying a conductive ink on a surface. Alternatively, "applying” can mean contacting a patient or tissue or organism with a device or system disclosed herein, or contacting a patient with an electric field or current.

[038] "BED" or "bioelectric device" is a LLMC or LLEF system as disclosed herein.

[039] "Biofilm” is any group of microorganisms in which cells adhere to each other, for example on a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm extracellular polymeric substance, which is also referred to as slime (although not everything described as slime is a biofilm), is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial and hospital settings. The microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, by contrast, are single-cells that may float or swim in a liquid medium. Many different bacteria form biofilms, including gram-positive (e.g. Bacillus spp, Listeria monocytogenes, Staphylococcus spp, and lactic acid bacteria, including Lactobacillus plantarum and Lactococcus lactis) and gram- negative species (e.g. Escherichia coli, or Pseudomonas aeruginosa). Biofilms are highly resistant to antibiotics. Consequently, very high and/or long-term doses are often required to eradicate biofilm-related infections. Biofilms are responsible for diseases, such as:

a. Otitis media- the most common acute ear infection in US children. b. Bacterial endocarditis- infection of the inner surface of the heart and its valves. c. Cystic fibrosis- a chronic disorder resulting in increased susceptibility to serious lung infections.

d. Legionnaire's disease- an acute respiratory infection resulting from the aspiration of clumps of Legionnella biofilms detached from air and water heating/cooling and distribution systems

e. Hospital-acquired infection- infections acquired from the surfaces of catheters, medical implants, wound dressing, or other medical devices.

f. Kidney stones- Biofilms also cause the formation of kidney stones. The stones cause symptoms of disease by obstructing urine flow and by producing inflammation and recurrent infection that can lead to kidney failure. Approximately 15%-20% of kidney stones occur in the context of a urinary tract infection. These stones can be produced by the interplay between infecting bacteria and mineral substrates derived from the urine. This interaction results in a complex biofilm composed of bacteria, bacterial exoproducts, and mineralized stone material.

g. Leptospirosis- Biofilms also cause leptospirosis, a serious but neglected emerging disease that infects humans through contaminated water. Previously, scientists believed the bacteria associated with leptospirosis were planktonic (free-floating). One research team has shown that Leptospira interrogans can make biofilms, which could be one of the main factors controlling survival and disease transmission. According to the study's author, 90% of the species of Leptospira tested could form biofilms, and it takes L interrogans an average of 20 days to make a biofilm.

h. Osteomyelitis- Biofilms may also cause osteomyelitis, a disease in which the bones and bone marrow become infected. This is supported by the fact that microscopy studies have shown biofilm formation on infected bone surfaces from humans and experimental animal models.

i. Osteonecrosis and osteomyelitis of the jaw- Many patients with these bone diseases exhibit large surface areas of bone occluded with well-developed biofilms.

j. Periodontal disease- Perhaps the most well-known and studied biofilm bacteria. Hundreds of microbial biofilm colonize the human mouth, causing tooth decay and gum disease.

k. Biofilms can be formed by bacteria that colonize plants, e.g. Pseudomonas putida, Pseudomonas fluorescens, and related pseudomonads which are common plant-associated bacteria found on leaves, roots, and in the soil, and the majority of their natural isolates form biofilms. Several nitrogen-fixing symbionts of legumes such as Rhizobium leguminosarum and Sinorhizobium meliloti form biofilms on legume roots and other inert surfaces can be used.

[040] "Conductive material” as used herein refers to an object or type of material which permits the flow of electric charges in one or more directions. Conductive materials can include solids such as metals or carbon, or liquids such as conductive metal solutions and conductive gels. Conductive materials can be applied to form at least one matrix. Conductive liquids can dry, cure, or harden after application to form a solid material.

[041] "Discontinuous region” as used herein refers to a "void” in a material such as a hole, slot, or the like. The term can mean any void in the material though typically the void is of a regular shape. The void in the material can be entirely within the perimeter of a material or it can extend to the perimeter of a material.

[042] "Dots” as used herein refers to discrete deposits of dissimilar reservoirs that can function as at least one battery cell. The term can refer to a deposit of any suitable size or shape, such as squares, circles, triangles, lines, etc. The term can be used synonymously with, microcells, etc.

[043] "Electrode” refers to similar or dissimilar conductive materials. In embodiments utilizing an external power source the electrodes can comprise similar conductive materials. In embodiments that do not use an external power source, the electrodes can comprise dissimilar conductive materials that can define an anode and a cathode.

[044] "Expandable” as used herein refers to the ability to stretch while retaining structural integrity and not tearing. The term can refer to solid regions as well as discontinuous or void regions; solid regions as well as void regions can stretch or expand.

[045] "Matrix” or "matrices” as used herein refer to a pattern or patterns, such as those formed by electrodes on a surface. Matrices can be designed to vary the electric field or electric microcurrent generated. For example, the strength and shape of the field or microcurrent can be altered, or the matrices can be designed to produce an electric field(s) or current of a desired strength or shape.

[046] "Stretchable” as used herein refers to the ability of embodiments that stretch without losing structural integrity. That is, embodiments can stretch to accommodate irregular wound surfaces or surfaces wherein one portion of the surface can move relative to another portion.

[047] LLMC / LLEF Systems- Methods of Manufacture

[048] Embodiments of the LLMC or LLEF systems disclosed herein can comprise electrodes or microcells, for example, on a substrate such as a bandage, tubing such as capillary tubing, a filter, or the like. Each electrode or microcell can be or include a conductive metal. In embodiments, the electrodes or microcells can comprise any electrically-conductive material, for example, an electrically conductive hydrogel, metals, electrolytes, superconductors, semiconductors, plasmas, and nonmetallic conductors such as graphite and conductive polymers. Electrically conductive metals can include silver, copper, gold, aluminum, molybdenum, zinc, lithium, tungsten, brass, carbon, nickel, iron, palladium, platinum, tin, bronze, carbon steel, lead, titanium, stainless steel, mercury, Fe/Cr alloys, and the like. The electrode can be coated or plated with a different metal such as aluminum, gold, platinum or silver.

[049] In certain embodiments, reservoir or electrode or cell geometry can comprise circles, polygons, lines, zigzags, ovals, stars, or any suitable variety of shapes. This provides the ability to design/customize surface electric field shapes as well as depth of penetration.

[050] Reservoir or dot sizes and concentrations can be of various sizes, as these variations can allow for changes in the properties of the electric field created. Certain embodiments provide an electric field at about 1 Volt and then, under normal tissue loads with resistance of 100k to 300K ohms, produce a current in the range of 10 microamperes. The electric field strength can be determined by calculating ½ the separation distance and applying it in the z- axis over the midpoint between the cell. This indicates the theoretical location of the highest strength field line.

[051] In embodiments "ink" or "paint" can comprise any conductive solution suitable for forming an electrode on a surface, such as a conductive metal solution. In embodiments "printing” or "painted" can comprise any method of applying a conductive material such as a conductive liquid material to a material upon which a matrix is desired.

[052] In embodiments printing devices can be used to produce LLMC or LLEF systems disclosed herein. For example, inkjet or 3D printers can be used to produce embodiments.

[053] In certain embodiments the binders or inks used to produce LLMC or LLEF systems disclosed herein can include, for example, poly cellulose inks, poly acrylic inks, poly urethane inks, silicone inks, and the like. In embodiments the type of ink used can determine the release rate of electrons from the reservoirs. In embodiments various materials can be added to the ink or binder such as, for example, conductive or resistive materials can be added to alter the shape or strength of the electric field. Other materials, such as silicon, can be added to enhance scar reduction. Such materials can also be added to the spaces between reservoirs.

[054] The power source can be any energy source capable of generating a current in the LLMC system and can include, for example, AC power, DC power, radio frequencies (RF) such as pulsed RF, induction, ultrasound, and the like. Certain embodiments can utilize a power source to create the electric current, such as a battery or a microbattery.

[055] Dissimilar metals used to make a LLMC or LLEF system disclosed herein can be silver and zinc, and the electrolytic solution can include sodium chloride in water. A preferred material to use in combination with silver to create the voltaic cells or reservoirs of disclosed embodiments is zinc. Zinc has been well-described for its uses in prevention of infection in such topical antibacterial agents as Bacitracin zinc, a zinc salt of Bacitracin. Zinc is a divalent cation with antibacterial properties of its own in addition to possessing the added benefit of being a cofactor to proteins of the metalloproteinase family of enzymes important to the phagocytic debridement and remodeling phases of wound healing. As a cofactor zinc promotes and accelerates the functional activity of these enzymes, resulting in better more efficient wound healing.

[056] Turning to the figures, in FIG. 1 , the dissimilar electrodes first electrode 6 and second electrode 10 are applied onto a desired primary surface 2 of an article 4. In one embodiment primary surface is a surface of a LLMC or LLEF system that comes into direct contact with an area to be treated such as skin surface or a wound. In alternate embodiments primary surface 2 is one which is desired to be antimicrobial.

[057] In embodiments, LLMC systems can produce a low level micro-current of between for example about 1 and about 400 micro-amperes, between about 20 and about 380 micro- amperes, between about 400 and about 360 micro-amperes, between about 60 and about 340 micro-amperes, between about 80 and about 320 micro-amperes, between about 100 and about 300 micro-amperes, between about 120 and about 280 micro-amperes, between about 140 and about 260 micro-amperes, between about 160 and about 240 micro-amperes, between about 180 and about 220 micro-amperes, or the like.

[058] The applied electrodes or reservoirs or dots can adhere or bond to the desired primary surface 2 because a biocompatible binder is mixed, in embodiments into separate mixtures, with each of the dissimilar metals that will create the pattern of voltaic cells, in embodiments. Most inks are simply a carrier, and a binder mixed with pigment. Similarly, conductive metal solutions can be a binder mixed with a conductive element. The resulting conductive metal solutions can be used with an application method such as screen printing to apply the electrodes to the primary surface in predetermined patterns. Once the conductive metal solutions dry and/or cure, the patterns of spaced electrodes can substantially maintain their relative position, even on a flexible material such as that used for a LLMC or LLEF system.

[059] The binder can include any biocompatible liquid material that can be mixed with a conductive element (preferably metallic crystals of silver or zinc) to create a conductive solution which can be applied as a thin coating to a surface. One suitable binder is a solvent reducible polymer, such as the polyacrylic non-toxic silk-screen ink manufactured by COLORCON ^® Inc., a division of Berwind Pharmaceutical Services, Inc. (see COLORCON ^® NO-TOX ^® product line, part number NT28). In an embodiment the binder is mixed with high purity (at least 99.999%) metallic silver crystals to make the silver conductive solution. In further embodiments the crystals can be of lower purity, for example 99%, or 97%, or 96%, or 95%, or 93%, or 90%, or 88%, or lower.

[060] The silver crystals, which can be made by grinding silver into a powder, are preferably smaller than 100 microns in size or about as fine as flour. In an embodiment, the size of the crystals is about 325 mesh, which is typically about 40 microns in size or a little smaller. The binder is separately mixed with high purity (at least 99.99%, in an embodiment) metallic zinc powder which has also preferably been sifted through standard 325 mesh screen, to make the zinc conductive solution. For better quality control and more consistent results, most of the crystals used should be larger than 325 mesh and smaller than 200 mesh. For example the crystals used should be between 200 mesh and 325 mesh, or between 210 mesh and 310 mesh, between 220 mesh and 300 mesh, between 230 mesh and 290 mesh, between 240 mesh and 280 mesh, between 250 mesh and 270 mesh, between 255 mesh and 265 mesh, or the like.

[061] Other powders of metal can be used to make other conductive metal solutions in the same way as described in other embodiments.

[062] When COLORCON ^® polyacrylic ink is used as the binder, about 10 to 40 percent of the mixture should be metal for a longer term bandage (for example, one that stays on for about 10 days). For example, for a longer term LLMC or LLEF system the percent of the mixture that should be metal can be 8 percent, or 10 percent, 12 percent, 14 percent, 16 percent, 18 percent. 20 percent, 22 percent, 24 percent, 26 percent, 28 percent. 30 percent, 32 percent, 34 percent, 36 percent, 38 percent, 40 percent, 42 percent, 44 percent, 46 percent, 48 percent, 50 percent, or the like. In embodiments a polycellulose ink can be used as a binder.

[063] To maximize the number of voltaic cells, in various embodiments, a pattern of alternating silver masses or electrodes or reservoirs and zinc masses or electrodes or reservoirs can create an array of electrical currents across the primary surface. A basic pattern, shown in FIG. 1 , has each mass of silver equally spaced from four masses of zinc, and has each mass of zinc equally spaced from four masses of silver, according to an embodiment. The first electrode 6 is separated from the second electrode 10 by a spacing 8. The designs of first electrode 6 and second electrode 10 are simply round dots, and in an embodiment, are repeated. Numerous repetitions 12 of the designs result in a pattern. For a wound management system or dressing, each silver design preferably has about twice as much mass as each zinc design, in an embodiment. For the pattern in FIG. 1 , the silver designs are most preferably about a millimeter from each of the closest four zinc designs, and vice- versa. The resulting pattern of dissimilar metal masses defines an array of voltaic cells when introduced to an electrolytic solution.

[064] A dot pattern of masses like the alternating round dots of FIG. 1 can be preferred when applying conductive material onto a flexible material, such as those used for a wound dressing, because the dots won't significantly affect the flexibility of the material. The pattern of FIG. 1 is well suited for general use. To maximize the density of electrical current over a primary surface the pattern 14 of FIG. 2 can be used. The first electrode 6 in FIG. 2 is a large hexagonally shaped dot, and the second electrode 10 is a pair of smaller hexagonally shaped dots that are spaced from each other. The spacing 8 that is between the first electrode 6 and the second electrode 10 maintains a relatively consistent distance between adjacent sides of the designs. Numerous repetitions 12 of the designs result in a pattern 14 that can be described as at least one of the first design being surrounded by six hexagonally shaped dots of the second design. The pattern 14 of FIG. 2 is well suited for abrasions and bums, as well as for insect bites, including those that can transfer bacteria or microbes or other organisms from the insect. There are of course other patterns that could be printed to achieve similar results.

[065] FIGS. 3 and 4 show how the pattern of FIG. 2 can be used to make an adhesive bandage. The pattern shown in detail in FIG. 2 is applied to the primary surface 2 of a wound dressing material. The back 20 of the printed dressing material is fixed to an absorbent wound dressing layer 22 such as cotton. The absorbent dressing layer is adhesively fixed to an elastic adhesive layer 16 such that there is at least one overlapping piece or anchor 18 of the elastic adhesive layer that can be used to secure the wound management system over a wound.

[066] FIG. 5 shows an additional feature, which can be added between designs, that will start the flow of current in a poor electrolytic solution. A fine line 24 is printed using one of the conductive metal solutions along a current path of each voltaic cell. The fine line will initially have a direct reaction but will be depleted until the distance between the electrodes increases to where maximum voltage is realized. The initial current produced is intended to help control edema so that the LLMC system will be effective. If the electrolytic solution is highly conductive when the system is initially applied the fine line can be quickly depleted and the wound dressing will function as though the fine line had never existed.

[067] FIGS. 6 and 7 show alternative patterns that use at least one line design. The first electrode 6 of FIG. 6 is a round dot similar to the first design used in FIG. 1. The second electrode 10 of FIG. 6 is a line. When the designs are repeated, they define a pattern of parallel lines that are separated by numerous spaced dots. FIG. 7 uses only line designs. The pattern of FIG. 7 is well suited for cuts, especially when the lines are perpendicular to a cut. The first electrode 6 can be thicker or wider than the second electrode 10 if the oxidation-reduction reaction requires more metal from the first conductive element (mixed into the first design's conductive metal solution) than the second conductive element (mixed into the second design's conductive metal solution). The lines can be dashed. Another pattern can be silver grid lines that have zinc masses in the center of each of the cells of the grid. The pattern can be letters printed from alternating conductive materials so that a message can be printed onto the primary surface-perhaps a brand name or identifying information such as patient blood type. [068] Because the spontaneous oxidation-reduction reaction of silver and zinc uses a ratio of approximately two silver to one zinc, the silver design can contain about twice as much mass as the zinc design in an embodiment. At a spacing of about 1 mm between the closest dissimilar metals (closest edge to closest edge) each voltaic cell that is in wound fluid can create approximately 1 Volt of potential that will penetrate substantially through the dermis and epidermis. Closer spacing of the dots can decrease the resistance, providing less potential, and the current will not penetrate as deeply. If the spacing falls below about one tenth of a millimeter a benefit of the spontaneous reaction is that which is also present with a direct reaction; silver is electrically driven into the wound, but the current of injury may not be substantially simulated. Therefore, spacing between the closest conductive materials can be 0.1 mm, or 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm,

2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3 mm, 3.1 mm, 3.2 mm,

3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4 mm, 4.1 mm, 4.2 mm, 4.3 mm,

4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, 5 mm, 5.1 mm, 5.2 mm, 5.3 mm, 5.4 mm,

5.5 mm, 5.6 mm, 5.7 mm, 5.8 mm, 5.9 mm. 6 mm, or the like.

[069] In certain embodiments spacing between the closest conductive materials can be not less than 0.1 mm, or not less than 0.2 mm, not less than 0.3 mm, not less than 0.4 mm, not less than 0.5 mm, not less than 0.6 mm, not less than 0.7 mm, not less than 0.8 mm, not less than 0.9 mm, not less than 1 mm, not less than 1.1 mm, not less than 1.2 mm, not less than

1.3 mm, not less than 1.4 mm. not less than 1.5 mm, not less than 1.6 mm. not less than 1.7 mm, not less than 1.8 mm, not less than 1.9 mm, not less than 2 mm, not less than 2.1 mm, not less than 2.2 mm, not less than 2.3 mm, not less than 2.4 mm, not less than 2.5 mm, not less than 2.6 mm, not less than 2.7 mm, not less than 2.8 mm, not less than 2.9 mm, not less than 3mm, not less than 3.1 mm, not less than 3.2 mm, not less than 3.3 mm, not less than

3.4 mm, not less than 3.5 mm, not less than 3.6 mm. not less than 3.7 mm, not less than 3.8 mm, not less than 3.9 mm, not less than 4 mm, not less than 4.1 mm, not less than 4.2 mm, not less than 4.3 mm, not less than 4.4 mm, not less than 4.5 mm, not less than 4.6 mm, not less than 4.7 mm, not less than 4.8 mm, not less than 4.9 mm, not less than 5 mm, not less than 5.1 mm, not less than 5.2 mm, not less than 5.3 mm, not less than 5.4 mm, not less than

5.5 mm, not less than 5.6 mm. not less than 5.7 mm, not less than 5.8 mm. not less than 5.9 mm, not less than 6 mm, or the like.

[070] Disclosures of the present specification include LLMC or LLEF systems comprising a primary surface of a pliable material wherein the pliable material is adapted to be applied to an area of tissue; a first electrode design formed from a first conductive liquid that includes a mixture of a polymer and a first element, the first conductive liquid being applied into a position of contact with the primary surface, the first element including a metal species, and the first electrode design including at least one dot or reservoir, wherein selective ones of the at least one dot or reservoir have approximately a 1.5 mm +/- 1 mm mean diameter; a second electrode design formed from a second conductive liquid that includes a mixture of a polymer and a second element, the second element including a different metal species than the first element, the second conductive liquid being printed into a position of contact with the primary surface, and the second electrode design including at least one other dot or reservoir, wherein selective ones of the at least one other dot or reservoir have approximately a 2.5 mm +/- 2 mm mean diameter; a spacing on the primary surface that is between the first electrode design and the second electrode design such that the first electrode design does not physically contact the second electrode design, wherein the spacing is approximately 1.5 mm +/- 1 mm, and at least one repetition of the first electrode design and the second electrode design, the at least one repetition of the first electrode design being substantially adjacent the second electrode design, wherein the at least one repetition of the first electrode design and the second electrode design, in conjunction with the spacing between the first electrode design and the second electrode design, defines at least one pattern of at least one voltaic cell for spontaneously generating at least one electrical current when introduced to an electrolytic solution. Therefore, electrodes, dots or reservoirs can have a mean diameter of 0.2 mm, or 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm,

2.4 mm, 2.5 mm„ 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm,

4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, 5.0 mm, or the like.

[071] The material concentrations or quantities within and/or the relative sizes (e.g., dimensions or surface area) of the first and second reservoirs can be selected deliberately to achieve various characteristics of the systems' behavior. For example, the quantities of material within a first and second reservoir can be selected to provide an apparatus having an operational behavior that depletes at approximately a desired rate and/or that "dies" after an approximate period of time after activation. In an embodiment the one or more first reservoirs and the one or more second reservoirs are configured to sustain one or more currents for an approximate pro-determined period of time, after activation. It is to be understood that the amount of time that currents are sustained can depend on external conditions and factors (e.g., the quantity and type of activation material), and currents can occur intermittently depending on the presence or absence of activation material.

[072] In various embodiments the difference of the standard potentials of the first and second reservoirs can be in a range from 0.05 V to approximately 5.0 V. For example, the standard potential can be 0.05 V, or 0.06 V, 0.07 V, 0.08 V, 0.09 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1.0 V, 1.1 V, 1.2 V, 1.3 V, 1.4 V, 1.5 V, 1.6 V, 1.7 V, 1.8 V, 1.9 V, 2.0 V, 2.1 V, 2.2 V, 2.3 V, 2.4 V, 2.5 V, 2.6 V, 2.7 V, 2.8 V, 2.9 V, 3.0 V, 3.1 V, 3.2 V, 3.3 V, 3.4 V, 3.5 V, 3.6 V, 3.7 V, 3.8 V, 3.9 V, 4.0 V, 4.1 V, 4.2 V, 4.3 V, 4.4 V, 4.5 V, 4.6 V, 4.7 V, 4.8 V, 4.9 V, 5.0 V, 5.1 V, 5.2 V, 5.3 V, 5.4 V, 5.5 V, 5.6 V, 5.7 V, 5.8 V, 5.9 V, 6.0 V, or the like.

[073] The voltage present at the site of treatment is typically in the range of millivolts but disclosed embodiments can introduce a much higher voltage, for example near 1 volt when using the 1 mm spacing of dissimilar metals already described. Further, in embodiments utilizing AC power the voltage introduced can vary or cycle over time. In embodiments the higher voltage can drive the current deeper into the treatment area so that dermis and epidermis benefit from the simulated current of injury. In this way the current not only can drive silver and zinc into the treatment area, but the current can also provide a stimulatory current so that the entire surface area can heal simultaneously. In embodiments the current can, for example, kill microbes. In embodiments the electric field can, for example, kill microbes.

[074] Embodiments disclosed herein relating to treatment of diseases or conditions or symptoms can also comprise selecting a patient or tissue in need of, or that could benefit by, treatment of that disease, condition, or symptom.

[075] While various embodiments have been shown and described, it will be realized that alterations and modifications can be made thereto without departing from the scope of the following claims. For example it can be desirable to use methods other than a common screen printing machine to apply the electrodes onto surfaces on medical instruments, garments, implants and the like so that they are antimicrobial. It is expected that other methods of applying the conductive material can be substituted as appropriate. Also, there are numerous shapes, sizes and patterns of voltaic cells that have not been described but it is expected that this disclosure will enable those skilled in the art to incorporate their own designs which will then be applied to a surface to create voltaic cells which will become active when brought into contact with an electrolytic solution.

[076] Certain embodiments include LLMC or LLEF systems comprising dressings or bandages designed to be used on irregular, non-planar, or "stretching” surfaces such as joints. Embodiments disclosed herein can be used with numerous joints of the body, including the jaw, the shoulder, the elbow, the wrist, the finger joints, the hip, the knee, the ankle, the toe joints, etc. Additional embodiments disclosed herein can be used in areas where tissue is prone to movement, for example the eyelid, the ear, the lips, the nose, genitalia, etc.

[077] Certain embodiments disclosed herein include a method of manufacturing a substantially planar LLMC or LLEF system, the method comprising joining with a substrate multiple first reservoirs wherein selected ones of the multiple first reservoirs include a reducing agent, and wherein first reservoir surfaces of selected ones of the multiple first reservoirs are proximate to a first substrate surface; and joining with the substrate multiple second reservoirs wherein selected ones of the multiple second reservoirs include an oxidizing agent, and wherein second reservoir surfaces of selected ones of the multiple second reservoirs are proximate to the first substrate surface, wherein joining the multiple first reservoirs and joining the multiple second reservoirs comprises joining using tattooing. In embodiments the substrate can comprise gauzes comprising dots or electrodes.

[078] Further embodiments can include a method of manufacturing a LLMC or LLEF system, the method comprising joining with a substrate multiple first reservoirs wherein selected ones of the multiple first reservoirs include a reducing agent, and wherein first reservoir surfaces of selected ones of the multiple first reservoirs are proximate to a first substrate surface; and joining with the substrate multiple second reservoirs wherein selected ones of the multiple second reservoirs include an oxidizing agent, and wherein second reservoir surfaces of selected ones of the multiple second reservoirs are proximate to the first substrate surface, wherein joining the multiple first reservoirs and joining the multiple second reservoirs comprises: combining the multiple first reservoirs, the multiple second reservoirs, and multiple parallel insulators to produce a pattern repeat arranged in a first direction across a plane, the pattern repeat including a sequence of a first one of the parallel insulators, one of the multiple first reservoirs, a second one of the parallel insulators, and one of the multiple second reservoirs; and weaving multiple transverse insulators through the first parallel insulator, the one first reservoir, the second parallel insulator, and the one second reservoir in a second direction across the plane to produce a woven apparatus.

[079] Further embodiments can comprise liquid-holding devices comprising a multi-array matrix of biocompatible microcells to apply an electric field, an electric current, or both to a fluid. For example, disclosed embodiments can comprise a "ring" or "sleeve" comprising a multi-array matrix of biocompatible microcells to apply an electric field, an electric current, or both to a fluid.

[080] LLMC / LLEF Systems- Methods of Use

[081] Disclosed embodiments comprise passing a bodily fluid over or past a substrate comprising electrodes, such as a bandage, tubing such as capillary tubing, a filter, or the like. Further embodiments comprise applying a substrate comprising electrodes to an area of the body.

[082] Treatment of Bacterial I nfections

[083] Embodiments of the disclosed LLMC and LLEF systems can provide antimicrobial activity. For example, embodiments disclosed herein can prevent, limit, or reduce formation of biofilms by interfering with bacterial signaling. Further embodiments can kill bacteria through an established biofilm.

[084] Embodiments comprise administration of an antibacterial combined with application of an electrical field or current to a patient. For example, a patient can be administered an oral antibacterial and then an electrical field or current can be applied to all or part of the patient's body.

[085] In embodiments, a fluid such as blood or plasma is removed from the patient, exposed to an electric field or current, then returned to the patient. In embodiments, a fluid such as blood or plasma is exposed to an electric field or current, then administered to the patient.

[086] Modulation of Enzyme Activity

[087] Methods and devices disclosed herein can be used to modulate enzyme activity. For example, embodiments can modulate the activity enzymes that are affected by electric fields or electric currents or both. For example, embodiments disclosed herein can modulate the activity of oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, and the like.

[088] In an embodiment, the activity of glycerol-3-phosphate dehydrogenase can be modulated.

[089] In embodiments, methods and devices disclosed herein can modulate the activity of co-enzymes.

[090] Embodiments can be used to modulate, for example, enzyme activity in mammalian, bacterial, insect, or other cells.

[091] Modulation of Gene Expression

[092] Methods and devices disclosed herein can be used to modulate gene expression. For example, methods and devices disclosed herein can be used for reducing expression of quorum sensing genes such as lasR and rhIR. Further embodiments can reduce expression of genes of the redox sensing multidrug efflux system, for example mexAB and mexEF. Embodiments can be used to modulate, for example, gene expression in mammalian, bacterial, insect, or other cells.

EXAMPLES

[093] The following non-limiting examples are provided for illustrative purposes only in order to facilitate a more complete understanding of representative embodiments. These examples should not be construed to limit any of the embodiments described in the present specification including those pertaining to the methods of treating wounds.

Example 1

Modulation of bacterial gene expression and enzvme activity

[094] Treatment of biofilms presents a major challenge, because bacteria living within them enjoy increased protection against host immune responses and are markedly more tolerant to antibiotics. Bacteria residing within biofilms are encapsulated in an extracellular matrix, consisting of several components including polysaccharides, proteins and DNA which acts as a diffusion barrier between embedded bacteria and the environment thus retarding penetration of antibacterial agents. Additionally, due to limited nutrient accessibility, the biofilm-residing bacteria are in a physiological state of low metabolism and dormancy increasing their resistance towards antibiotic agents.

[095] Chronic wounds present an increasing socio-economic problem and an estimated 1- 2% of western population suffers from chronic ulcers and approximately 2-4% of the national healthcare budget in developed countries is spent on treatment and complications due to chronic wounds. The incidence of non-healing wounds is expected to rise as a natural consequence of longer lifespan and progressive changes in lifestyle like obesity, diabetes, and cardiovascular disease. Non-healing skin ulcers are often infected by biofilms. Multiple bacterial species reside in chronic wounds; with Pseudomonas aeruginosa, especially in larger wounds, being the most common. P. aeruginosa is suspected to delay healing of leg ulcers. Also, surgical success with split graft skin transplantation and overall healing rate of chronic venous ulcers is presumably reduced when there is clinical infection by P. aeruginosa.

[096] P. aeruginosa biofilm is often associated with chronic wound infection. The BED ("BED" or "bioelectric device " or PROCELLERA ^® as disclosed herein) consists of a matrix of silver-zinc coupled biocompatible microcells, which in the presence of conductive wound exudate activates to generate an electric field (0.3- 0.9V). Growth (measured as O.D and cfu) of pathogenic Pseudomonas aeruginosa strain PAO1 in LB media was markedly arrested in the presence of the BED (p<0.05, n=4). PAO1 biofilm was developed in vitro using a polycarbonate filter model. Grown overnight in LB medium at 37°C bacteria were cultured on sterile polycarbonate membrane filters placed on LB agar plates and allowed to form a mature biofilm for 48h. The biofilm was then exposed to BED or placebo for the following 24h. Structural characterization using scanning electron microscopy demonstrated that the BED markedly disrupted biofilm integrity as compared to no significant effect observed using a commercial silver dressing commonly used for wound care. Staining of extracellular polymeric substance, PAO1 staining, and a vital stain demonstrated a decrease in biofilm thickness and number of live bacterial cells in the presence of BED (n=4). BED repressed the expression of quorum sensing genes lasR and rhIR (p<0.05, n=3). BED was also found to generate micromolar amounts of superoxide (n=3), which are known reductants and repress genes of the redox sensing multidrug efflux system mexAB and mexEF (n=3, p<0.05). BED also down- regulated the activity of glycerol-3-phosphate dehydrogenase, an electric field sensitive enzyme responsible for bacterial respiration, glycolysis, and phospholipid biosynthesis (p<0.05, n=3).

[097] Materials and Methods

[098] In-vitro biofilm model

[099] PAO1 biofilm was developed in vitro using a polycarbonate filter model. Cells were grown overnight in LB medium at 37°C bacteria were cultured on sterile polycarbonate membrane filters placed on LB agar plates and allowed to form a mature biofilm for 48h. The biofilm was then exposed to BED or placebo for the following 24h.

[0100] Energy Dispersive X-rav Spectroscopy (EDS)

[0101] EDS elemental analysis of the Ag/ZN BED was performed in an environmental scanning electron microscope (ESEM, FEI XL-30) at 25kV. A thin layer of carbon was evaporated onto the surface of the dressing to increase the conductivity.

[0102] Scanning electron microscopy

[0103] Biofilm was grown on circular membranes and was then fixed in a 4% formaldehyde/2% glutaraldehyde solution for 48 hours at 4°C, washed with phosphate- buffered saline solution buffer, dehydrated in a graded ethanol series, critical point dried, and mounted on an aluminum stub. The samples were then sputter coated with platinum (Pt) and imaged with the SEM operating at 5 kV in the secondary electron mode (XL 30S; FEG, FEI Co., Hillsboro, OR).

[0104] Live/Dead staining

[0105] The LIVE/DEAD BacLight Bacterial Viability Kit for microscopy and quantitative assays was used to monitor the viability of bacterial populations. Cells with a compromised membrane that are considered to be dead or dying stain red, whereas cells with an intact membrane stain green.

[0106] EPR spectroscopy

[0107] EPR measurements were performed at room temperature using a Bruker ER 300 EPR spectrometer operating at X-band with a TM 110 cavity. The microwave frequency was measured with an EIP Model 575 source-locking microwave counter (EIP Microwave, Inc., San Jose, CA). The instrument settings used in the spin trapping experiments were as follows: modulation amplitude, 0.32 G; time constant, 0.16 s; scan time, 60 s; modulation frequency, 100 kHz; microwave power, 20 mW; microwave frequency, 9.76 GHz. The samples were placed in a quartz EPR flat cell, and spectra were recorded at ambient temperature (25°C). Serial 1-min EPR acquisitions were performed. The components of the spectra were identified, simulated, and quantitated as reported. The double integrals of DEPMPO experimental spectra were compared with those of a 1 mM TEMPO sample measured under identical settings to estimate the concentration of superoxide adduct.

[0108] Quantification of mRNA and miRNA Expression

[0109] Total RNA, including the miRNA fraction, was isolated using Norgen RNA isolation kit, according to the manufacturer's protocol. Gene expression levels were quantified with real- time PCR system and SYBR Green (Applied Biosystems) and normalized to nadB and proC as housekeeping genes. Expression levels were quantified employing the 2 (-DD ) relative quantification method.

[0110] Glvcerol-3-phosphate dehydrogenase assay [0111] The Glycerol-3-phosphate dehydrogenase assay was performed using an assay kit from Biovision, Inc. following manufacturer's instructions. Briefly, cells (~1 x 10 ⁶ ) were homogenized with 200 pi ice cold GPDH Assay buffer for 10 minutes on ice and the supernatant was used to measure O.D. and GPDH activity calculated from the results.

[0112] Statistics

[0113] Control and treated samples were compared by paired / test. Student's / test was used for all other comparison of difference between means. P < 0.05 was considered significant.

[0114] Ag/Zn BED disrupts P. aeruginosa biofilm

[0115] To validate the chemical composition of the dressing, we collected high resolution electron micrographs using an environmental scanning electron microscope. Our element maps indicate that silver particles are concentrated in the golden dots of the polyester cloth, while zinc particles are concentrated in the grey dots.

[0116] As illustrated in Figure 9A, P.aeruginosa was grown in round bottom tubes in LB medium with continuous shaking and absorbance was measured by calculating optical density at 600nm at different time points. It was observed that Ag/Zn BED and the control dressing with equal amount of silver inhibited bacterial growth (n=4) (Figure 9B,C). When bacteria is grown in an agar plate with Ag/Zn BED dressing or placebo embedded in the agar, the zone of inhibition is clearly visible in the case of Ag/Zn BED thus demonstrating its bacteriostatic property, while placebo with silver dressing showed a smaller zone of inhibition, indicating the effect role of electric field as compared to topical contact. (Figure 9D). However, as evident from scanning electron microscope images (Figure 10); EPS staining (Figure 11); and live/dead staining (Figure 12), Ag/Zn BED disrupts biofilm much better while silver does not have any effect on biofilm disruption. Silver has been recognized for its antimicrobial properties for centuries. Most studies on the antibacterial efficacy of silver, with particular emphasis on wound healing, have been performed on planktonic bacteria. Silver ions, bind to and react with proteins and enzymes, thereby causing structural changes in the bacterial cell wall and membranes, leading to cellular disintegration and death of the bacterium. Silver also binds to bacterial DNA and RNA, thereby inhibiting the basal life processes.

[0117] Silver is effective against mature biofilms of P. aeruginosa, but only at a high silver concentration. A concentration of 5-10 mg/mL silver sulfadiazine has been reported to eradicate biofilm whereas a lower concentration (1 mg/mL) had no effect. Therefore, the concentration of silver in currently available wound dressings is much too low for treatment of chronic biofilm wounds. Figure 13 shows PAO1 staining of the biofilm demonstrating the lack of elevated mushroom like structures in the Ag/Zn BED treated sample.

[0118] Ag/Zn BED down regulates quorum sensing genes

[0119] The pathogenicity of P. aeruginosa is attributable to an arsenal of virulence factors. The production of many of these extracellular virulence factors occurs only when the bacterial cell density has reached a threshold (quorum). Quorum sensing is controlled primarily by two cell-to-cell signaling systems, called las and rhl, which are both composed of a transcriptional regulator (LasR and RhIR, respectively) and an autoinducer synthase (Lasl and Rhll, respectively). In P. aeruginosa, Lasl produces 3OC12-HSL LasR, then, responds to this signal and the LasR: 3OC12-HSL complex activates transcription of many genes including rhIR, which encodes a second quorum sensing receptor [26-30], RhIR which binds to autoinducer C4-HSL produced by Rhll. RhlR:C4- HSL also directs a large regulon of genes. P. aeruginosa defective in QS is compromised in their ability to form biofilms. Quorum sensing inhibitors increase the susceptibility of the biofilms to multiple types of antibiotics.

[0120] To test the effect of the electric field on quorum sensing genes, we subjected the mature biofilm to the Ag/Zen BED or placebo for 12 hours and looked at gene expression levels. We selected an earlier time point, because by 24 hours, as in earlier experiments, most bacteria under Ag/Zn BED treatment were dead. We found a significant down regulation of lasR and rhIR (n=4, p<0.05). lasR transcription has been reported to weakly correlate with the transcription of lasA, lasB, toxA and aprA. We did not, however, find any significant difference in their expression levels at this time point, although we found them down regulated in the Ag/Zn BED treated samples at the 24 hour time point (data not shown). (Figure 14).

[0121] Ag/Zn BED represses the redox sensing multidruo efflux system in P. aeruginosa

[0122] Ag/Zn BED acts as a reducing agent and reduces protein thiols. One electron reduction of dioxygen O ₂ , results in the production of superoxide anion. Molecular oxygen (dioxygen) contains two unpaired electrons. The addition of a second electron fills one of its two degenerate molecular orbitals, generating a charged ionic species with single unpaired electrons that exhibit paramagnetism. Superoxide anion, which can act as a biological reductant and can reduce disulfide bonds, is finally converted to hydrogen peroxide is known to have bactericidal properties. In this paper, we use electron paramagnetic resonance (ERR) to detect superoxide directly upon exposure to the bioelectric dressing. Superoxide spin trap was carried out using DEPMPO (2-(diethoxyphosphoryi)-2-methyl-3,4-dihydro-2H-pyrrole 1- oxide) and ~1 mM superoxide anion production was detected upon 40 mins of exposure to Ag/Zn BED (Figure 15). MexR and MexT are two multidrug efflux regulators in P. aeruginosa which uses an oxidation-sensing mechanism. Oxidation of both MexR and MexT results in formation of intermolecular disulfide bonds, which activates them, leading to dissociation from promoter DNA and de-repression of MexAB-oprM and MexEF-oprN respectively, while in a reduced state, they do not transcribe the operons. Induction of Mex operons leads not only to increased antibiotic resistance but also to repression of the quorum sensing cascades and several virulence factors. We observe down-regulation of the downstream Mex genes MexA, MexB, Mex E and MexF (but not MexC and MexD) (n=4, p<0.05), in Ag/Zn BED treated samples, inactive forms of MexR and MexT in their reduced states. To confirm the reducing activity of the Ag/Zn BED, the experiments were repeated with 10mM DTT and similar results were observed. (Figure 16).

[0123] Ag/Zn BED diminishes alvcerol-3-phosphate dehydrogenase enzvme activity

[0124] Electric fields can affect molecular charge distributions on many enzymes. Glycerol- 3-phosphate dehydrogenase is an enzyme involved in respiration, glycolysis, and phospholipid biosynthesis and is expected to be influenced by external electric fields in P. aeruginosa. We observed significantly diminished glycerol-3-phosphate dehydrogenase enzyme activity by treating P. aeruginosa biofilm to the Ag/Zn BED for 12 hours (n=3).

Example 2

LLMC influence on biofilm properties

[0125] In this study ten clinical wound pathogens associated with chronic wound infections were used for evaluating the anti-biofilm properties of a LLMC. Hydrogel and drip-flow reactor (DFR) biofilm models were employed for the efficacy evaluation of the wound dressing in inhibiting biofilms. Biofilms formed with Acinetobacter baumannii, Corynebacterium amycolatum, Escherichia coli, Enterobacter aerogenes, Enterococcus faecaiis Cl 4413, Klebsiella pneumonia, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, and Streptococcus equi clinical isolates were evaluated. For antimicrobial susceptibility testing of biofilms, 10 ⁵ CFU/mL bacteria was used in both biofilm models. For poloxamer hydrogel model, the LLMCs (25 mm diameter) were applied directly onto the bacterial biofilm developed onto 30% poloxamer hydrogel and Muller-Hinton agar plates, and incubated at 37°C for 24 h to observe any growth inhibition. In the DFR biofilm model, bacteria were deposited onto polycarbonate membrane as abiotic surface, and sample dressings were applied onto the membrane. The DFR biofilm was incubated in diluted trypticase soy broth (TSB) at room temperature for 72 h. Biofilm formations were evaluated by crystal violet staining under light microscopy, and anti-biofilm efficacy was assessed by reduction in bacterial numbers.

Example 3

Modulation of mammalian gene expression and enzvme activity

[0126] Grown overnight in LB medium at 37°C, primary human dermal fibroblasts are cultured on sterile polycarbonate membrane filters placed on LB agar plates for 48h. The cells are then exposed to BED or placebo for the following 24h. BED represses the expression of glyceraldehyde 3-phosphate dehydrogenase. BED also down-regulated the activity of glyceraldehyde 3-phosphate dehydrogenase.

Example 4

Modulation of insect oene expression and enzvme activity

[0127] Grown overnight in LB medium at 37°C, drosophila S2 cells are cultured on sterile polycarbonate membrane filters placed on LB agar plates for 48h. The cells are then exposed to BED or placebo for the following 24h. BED represses the expression of insect P450 enzymes. BED also down-regulated the activity of insect P450 enzymes.

Example 5

LLMC influence on antibiotic-resistant bacteria

[0128] Disclosed embodiments were tested against antibiotic resistant bacteria.

[0129] Bacterial strains tested;

a. Multidrug-resistant Pseudomonas spp.

b. Carbapenem-resistant Acinetobacter spp.

c. Methicillin-resistant Staphylococcus aureus (MRSA).

d. Carbapenem-resistant Enterobacteriaceae (CRE)- Klebsiella pneumoniae subsp pneumonia.

[0130] The primary efficacy endpoint is the decrease or complete inhibition of bacterial viability after 24-48h of treatment. Results are shown in FIG'S 18-23 ( "WED” refers to an embodiment disclosed herein).

Example 6

LLMC in treatment of infection

[0131] A patient suffering from a systemic infection undergoes a treatment as described herein. Blood is removed from the patient, passed over an electric field, then returned to the patient.

Example 7

LLMC in treatment of infection

[0132] A patient suffering from a systemic infection undergoes a treatment as described herein. Blood is passed over an electric field produced by electrodes on a substrate, then administered to the patient.

Example 8

LLMC in treatment of infection

[0133] A patient suffering from a systemic infection undergoes a treatment as described herein. Blood is removed from the patient, passed over an electric field comprising capillary tubing comprising electrodes, then returned to the patient.

[0134] In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular methodology, protocol, and/or reagent, etc., described herein. As such, various modifications or changes to or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the present specification. Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. Accordingly, embodiments of the present disclosure are not limited to those precisely as shown and described.

[0135] Certain embodiments are described herein, including the best mode known to the inventor for carrying out the methods and devices described herein. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0136] Groupings of alternative embodiments, elements, or steps of the present disclosure are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0137] Unless otherwise indicated, all numbers expressing a characteristic, item, quantity, parameter, property, term, and so forth used in the present specification and claims are to be understood as being modified in all instances by the term "about." As used herein, the term "about" means that the characteristic, item, quantity, parameter, property, or term so qualified encompasses a range of plus or minus ten percent above and below the value of the stated characteristic, item, quantity, parameter, property, or term. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the disclosure are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numerical ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the present specification as if it were individually recited herein.

[0138] The terms "a,” "an," "the ^* and similar referents used in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope otherwise claimed. No language in the present specification should be construed as indicating any non-claimed element essential to the practice of embodiments disclosed herein.

[0139] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term‘consisting of excludes any element, step, or ingredient not specified in the claims. The transition term‘consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the present disclosure so claimed are inherently or expressly described and enabled herein.